SIRT2 downregulated in neuro2a cells and mice brains upon JEV infection.

(A) The representative confocal images of JE virus (one MOI) or mock-infected neuro2a cells at 30 hpi; JEV-NS3 protein stained in green, and the nuclei stained by Hoechst 33342 in blue. The scale bar =10 mM. (B) The virus load upon 1 MOI of JE virus infection in neuro2a cells over time expressed in plaque-forming units (PFUs). n=3 independent experiments; each experiment performed by duplicates. (C) Representative immunoblot image displaying a reduction in SIRT2 levels in neuro2a cells upon 1 MOI of JE virus infection at 30 hpi. The JEV protein NS5 confirms JEV infection in the cells. n=3 independent experiments; each experiment performed by duplicates. (D-I) 10-day-old mice were infected through the JE virus of 105 PFU by the foot-pad route of inoculation; n=15-16 mice per group. Graphical representation depicting the percentage of mock or JE virus-infected mice expressing clinical signs (D) and paralysis (E) at various dpi. (F) The percentage of mice surviving upon JEV mice at various dpi up to 24th dpi, in Kaplan-Meier survival percentage curve (n=15-16 mice each group). The p-value was calculated using the Log-rank (Mantel-Cox) test. Graphically representing the JEV load (PFUs) in the serum (G) and brain (H) of wild-type mice at various dpi; n=3 mice per dpi. (I) Representative immunoblot indicating the reduction in SIRT2 levels upon JEV-infection on 8th dpi. The detection of NS5 confirms the virus infection. n=6 mice in each group.

SIRT2 genetic deficiency increases JEV multiplication in neuro2a cells and mice brains.

(A) Representative western blotting confirms SIRT2 knock-down in neuro2a cells by siRNA. (B) Graph displaying the viral PFU levels at the various hpi of control or SIRT2 knock-down neuro2a cells infected with JE-Virus. n=3 independent experiments; triplicates were used in each experiment. P values are calculated using Student’s t-test. (C-I) 10-day-old mice were infected through the JE virus of 105 PFU by the foot-pad route of inoculation. (C) Schematic representation of JEV administration in mice by foot-pad route. (D) Representative immuno-blotting validating the absence of SIRT2 in the SIRT2-/- (SIRT2-KO) mice brain; JE virus infection in the mice brain was confirmed by detecting JEV-NS5 protein. (E) The graph represents JE virus PFUs at various dpi in the serum of WT or SIRT2-KO mice infected with JE virus. n=3 mice per dpi. (F) The graph represents JE virus PFUs at various dpi in the brains of JE virus-infected WT or SIRT2-/- mice. n=5 mice per dpi. P values are calculated using Student’s t-test. (G) The graph represents the percentage of animals expressing clinical signs at various dpi in JE virus-infected WT or SIRT2-/- mice (n=15 mice per group). (H) The graph depicts the percentage of animals displaying paralysis in JE virus-infected WT or SIRT2-/- mice at various dpi (n=15 mice in each group). (I) The percentage of animals surviving in JE virus-infected WT or SIRT2-/- mice at various dpi up to 24th dpi, in the Kaplan-Meier survival percentage curve. n=15 mice in each group; the p-value was calculated using the Log-rank (Mantel-Cox) test. (J-N) 10-day-old mice were inoculated with JE virus (3×103 PFUs) by an intra-cerebral route of inoculations. (J) Schematic representation of JEV administration in mice by an intra-cerebral route. (K) The graph represents JEV PFUs at 6th dpi in WT or SIRT2-/- mice brains. n=5 mice per group; the p-value was calculated using Student’s t-test. (L) The graph represents the percentage of animals expressing clinical signs at various dpi in JE virus-infected WT or SIRT2-/- mice. n=12-16 mice in each group. (M) The graph depicts the percentage of animals displaying clinical paralysis at various dpi in JE virus-infected WT or SIRT2-/- mice (n=12-16 mice in each group). (N) The percentage of animals surviving at various dpi up to 24th dpi in JE virus-infected WT or SIRT2-/- mice in the Kaplan-Meier survival percentage curve. n=12-16 mice in each group; the p-value was calculated using the Log-rank (Mantel-Cox) test.

AGK2 treatment exacerbated clinical manifestation in JEV-infected mice through induction of autophagy.

(A) Graph displaying the viral PFU levels at the various hpi of Vehicle or AGK2 treated neuro2a cells infected with JE-Virus. n=3 independent experiments; triplicates were used in each experiment. P values are calculated using Student’s t-test. (B-F) 10-day-old WT mice were inoculated with JE virus of 105 PFUs by the foot-pad route; AGK2 treatment started from 1st dpi onwards for every day up to 24th dpi through the intraperitoneal (IP) route. (B) Graph representing the percent animals expressing clinical signs at various dpi in vehicle or AGK2 treated JE virus infected mice; n=15-18 mice per group. (C) Graph depicting the percent of animals displaying paralysis at various dpi in JE virus-infected mice upon vehicle or AGK2 treatment. n=15-18 mice per group. (D) The percentage of mice surviving after JE virus infection upon vehicle or AGK2 treatment at various dpi is depicted in the Kaplan-Meier survival curve. Statistical analysis was done using Log rank (Mantel-Cox) test; n=15-18 mice per group. Graph representing JE virus load in (E) serum (PFU/ml) (n=3 mice per dpi) and (F) brain (PFU/10mg) (n=5 mice per dpi), upon vehicle or AGK2 treatment in JE virus-infected mice at various dpi. The p-value was calculated using Student’s t-test. (G) Representative immunoblot displaying differences in autophagy markers in the brains of the vehicle or AGK2 treated mice at 8th dpi of JE virus infection (n=3 mice each group). (H-J) 10-day-old Wild-Type mice were inoculated with 3×103 PFUs of JEV virus by an intra-cerebral route of inoculation, and AGK2 treatment started from first dpi for every day up to 24th dpi by the IP route of inoculation; n=10-14 mice each group. Graph displaying the percent of animals showing (H) clinical signs and (I) paralysis, at various dpi in JE virus-infected mice upon vehicle or AGK2 treatment. (n=10-14 mice each group). (J) The survival of JE virus-infected mice treated with vehicle or AGK2 up to 24th dpi in the Kaplan-Meier survival percentage curve. The p-value was calculated using the Log-rank (Mantel-Cox) test. (n=10-14 mice per group)

SIRT2 gene therapy using the AAV vector protects against JEV infection in mice.

(A-E) 10-day-old wild-type mice were inoculated with 105 PFUs of JEV through the foot-pad route, and 1015 copies of AAV-null or AAV-SIRT2 treatment started from first dpi and 4th dpi through the IP route. (A) Representative immune-blot displaying changes in SIRT2 protein and autophagy markers in the brain of AAV-null or AAV-SIRT2 treated wild-type mice infected with JEV at 8th dpi (n=3 mice each group). (B) The graph represents JE virus PFUs in the brain of JEV-infected wild-type mice, with AAV-null or AAV-SIRT2 treatment; n=3 mice per dpi, respectively. The p-value was calculated using Student’s t-test. The graph represents the percentage of animals exhibiting (C) clinical signs and (D) paralysis in mock or JE virus-infected wild-type mice treated with AAV-null or AAV-SIRT2 at various dpi (n=6-8 mice in each group). (E) Kaplan-Meier survival curve indicating the survival percentage of mock or JE virus-infected wild-type mice, with AAV-null or AAV-SIRT2 treatment at various dpi up to 24th dpi (n=6-8 mice in each group). The p-value was calculated using the Log-rank (Mantel-Cox) test.

Hyperactivation of NF-κB transcriptional activity-dependent genes in SIRT2 knock-out mice upon JEV infection.

(A-J) Transcriptome was performed in the mice brains infected with JE virus of 105 PFUs by the foot-pad route. n=3 mice each group. (A) Clustergram representing the top hit KEGG pathway enrichment based on the genes significantly differentially expressed in the mRNA transcriptome of mock or JE virus infected WT mice brain. (B) Clustergram representing the top hit TRRUST Transcription Factors enrichment based on the genes significantly differentially expressed in the mRNA transcriptome of mock or JE virus infected WT mice brain. (C) Graph depicting top hit KEGG pathways enrichment based on the genes significantly upregulated in the transcriptome of JE virus infected mice brain compared to mock-infected WT mice. (D) Graph depicting top hit TRRUST Transcription Factors enrichment based on the genes significantly upregulated in the transcriptome of JE virus infected mice brain compared to mock-infected WT mice. (E) Graph showing top hit KEGG pathways enrichment based on the genes significantly down-regulated in the transcriptome of JE virus infected mice brain compared to mock-infected WT mice. (F) Graph depicting top hit TRRUST Transcription Factors enrichment based on the genes significantly down-regulated in the transcriptome of JE virus infected mice brain compared to mock-infected WT mice. (G) Heatmap representing differential expression profile of genes regulated by NF-κB in the transcriptome of mock or JE virus infected WT mice brain. (H) Volcano plot displaying differentially expressed profile of genes regulated by NF-κB transcriptional activity in the transcriptome of mock or JE virus-infected WT mice brain with log2 fold change ≥2 and p ≤0.05(n=3 mice each group). (I) Heatmap representing differential expression profile of genes regulated by NF-κB transcriptional activity in the transcriptome of JE virus-infected WT or SIRT2-/- mice brain. (J) Volcano plot displaying differential expression profile of genes regulated by NF-κB transcriptional activity in the transcriptome of JE virus-infected WT or SIRT2-/- mice brain with log2 fold change ≥2 and p ≤0.05. (K-N) Ten-day-old mice pups were infected through the foot-pad route with 105 PFUs of JE virus. Relative fold change in the (K) CD69; (L) IL23a; (M) CXCL10; and (N) CCL5 mRNA levels on 8th dpi of mock or JE virus-infected WT or SIRT2-/- mice brains; n=4 mice per group; P value calculated using Student’s t-test.

SIRT2 and NF-κB interaction mediated acetylation status of NF-κB regulates the JEV replication.

(A) Immunoprecipitation of p65 at 30 hpi with mock or JE virus (1 MOI) infected neuro2a cells demonstrating p65 and SIRT2 interaction. (B) NF-κB activity was assayed at 30 hpi using luciferase reporter in mock or JE virus (1 MOI) infected neuro2a cells (n=3 independent experiments, triplicates were used in each experiment; The p-value was calculated using Student’s t-test). Relative fold change in the luciferase reporter activity of NF-κB at 30 hpi of JE virus infection in (C) control or SIRT2-KD; (D) Ad-null or Ad-SIRT2; treated conditions; n=3 independent experiments, triplicates were used in each experiment; Statistical analysis using one-way ANOVA. (E) The network of signaling pathways represents differential expression genes that are regulated by NF-κB transcriptional activity in the WT or SIRT2-/- mice brain upon JEV infection (n=3 mice each group). The qPCR analysis representing the relative fold changes in the beclin-1 expression levels in (F) JE virus-infected (1 MOI) neuro2a cells; (G) JEV (1 MOI) infected control or SIRT2-KD neuro2a cells; and (H) JEV (1 MOI) infected Ad-null or Ad-SIRT2 treated neuro2a cells at 30 hpi (n=3 independent experiments, triplicates were used in each experiment; The p-value was calculated using (B and F) Student’s t-test and (C-D and G-H) one-way ANOVA. (I) Representative immuno-blotting images displaying differences in autophagy markers at 30 hpi in the JE virus-infected neuro2a cells. (n=3 independent experiments; duplicates were used in each experiment). (J) Representative immuno-blotting images displaying an increase in autophagy markers at 8 dpi in the JE virus-infected WT mice brain (n=6 mice in each group). (K) Representative immuno-blotting images displaying differences in autophagy markers at 30 hpi in the JE virus-infected, control or SIRT2-KD neuro2a cells (n=3 independent experiments; duplicates were used in each experiment). (L) Representative immuno-blotting images displaying change in autophagy markers at 30 hpi in the JE virus-infected, vehicle or AGK2-treated neuro2a cells (n=3 independent experiments; duplicates were used in each experiment). (M) Representative immuno-blotting images displaying differences in autophagy markers at 8 dpi in the WT or SIRT2-/- mice brains upon JEV infection (n=6 mice in each group). (N) Representative immuno-blotting images displaying changes in autophagy markers at 30 hpi in the JE virus-infected neuro2a cells upon Ad-null or Ad-SIRT2 treatment (n=3 independent experiments; duplicates were used in each experiment). Statistical analysis of all immune-blotting experiments was performed using Student’s t-test.

SIRT2 exerts the antiviral effect by inhibiting NF-κB transcriptional activity.

(A) Graph displaying NF-κB activity assessed at 30 hpi using luciferase reporter assay in mock or JE virus (1 MOI) infected neuro2a cells treated with vehicle or Bay-11. Results are expressed as fold change relative to the vehicle-treated mock control group. (n=3 independent experiments, triplicates were used in each experiment; p-values are calculated using one-way ANOVA. (B) Graph corresponding to changes in viral PFUs at 30 and 60 hpi in JE virus (1 MOI) infected neuro2a cells with vehicle or Bay-11 treatment. n=3 independent experiments, triplicates were used in each experiment; The p-value was calculated using Student’s t-test. (C) Graphical representations of NF-κB activity evaluated by luciferase reporter assays in JE virus (1 MOI) infected neuro2a cells treated with vehicle or Bay-11 in Control siRNA or SIRT2 specific siRNA transfected neuro2a cells; Results are depicted as fold change relative to vehicle-treated non-transfected control groups. n=3 independent experiments, triplicates were used in each experiment; Statistical analysis using one-way ANOVA. (D) Graph displaying changes in viral PFUs at 30 hpi in JEV infected neuro2a cells upon transfection with Control siRNA or SIRT2 specific siRNA and further treatment with vehicle or BAY-11. (n=3 independent experiments, triplicates were used in each experiment); p-values are calculated using one-way ANOVA. (E) Graphical representations of NF-κB activity evaluated by luciferase reporter assays in JE virus (1 MOI) infected neuro2a cells treated with vehicle or Bay-11 in Ad-null or Ad-SIRT2 treated neuro2a cells; Results are depicted as fold change relative to vehicle-treated non-transfected control groups. n=3 independent experiments, triplicates were used in each experiment; Statistical analysis using one-way ANOVA. (F) Graph displaying changes in viral PFUs at 30 hpi in JEV infected neuro2a cells upon treatment with Ad-null or Ad-SIRT2 and further treatment with vehicle or BAY-11. (n=3 independent experiments, triplicates were used in each experiment); p-values are calculated using one-way ANOVA.

SIRT2 regulates the NF-κB mediated antiviral activity in JE virus-infected mice.

(A-E) 10-day-old WT or SIRT2-/- mice were inoculated with 105 PFUs of JEV through the foot-pad route, and vehicle or BAY-11 treatment started from first dpi through the IP route for every day up to 24th dpi. The graph represents the percentage of WT or SIRT2-/- animals exhibiting (A) clinical signs (B) paralysis in JE virus-infected vehicle or BAY-11 treated mice at various dpi (n=10-15 mice in each group). (C) Kaplan-Meier survival curve indicating the survival percentage of JE virus-infected, WT or SIRT2-/- mice, with vehicle or BAY-11 treatment at various dpi up to 24th dpi (n=10-15 mice each group). The p-value was calculated using the Log-rank (Mantel-Cox) test. The graph represents JE virus PFUs in the (D) serum and (E) brain of infected WT or SIRT2-/- mice, with vehicle or BAY-11 treatment; n=3 and 5 mice per dpi, respectively. The p-value was calculated using Student’s t-test.